J.S. van der Hoeven

The Growth of Oral Bacteria on Saliva

The present experiments were aimed at studying the degradation of salivary glycoproteins by the oral microflora. To this end, S. sanguis I strain Ny476 and S. sanguis II (S. mitior) strain Ny581 were grown continuously in human-whole saliva. Under these conditions, the strains produced a variety of cell-associated hydrolytic activities, including glycosidases, exo- and endopeptidases, and esterases. S. sanguis II generally exhibited higher levels of enzyme activity than did S. sanguis I, in particular of neuraminidase that was produced only by S. sanguis II. In accordance, S. sanguis II had a higher cell yield and consumed a higher proportion of the sugars and sialic acid in the glycoproteins than did S. sanguis I. Interestinglv, S. sanguis I, which is devoid of neuraminidase, is known to have a lectin with specificity for sialic acid, whereas S. sanguis II has affinity for galactose residues in the glycoproteins. We propose that specific binding of glycoproteins by oral bacteria constitutes a mechanism to collect nutrients in the vicinity of the cell. The special ability of S. sanguis II to utilize saliva for growth was further exemplified by its selection in batch-wise enrichments of dental plaque on saliva. The microflora in these enrichment cultures always consisted of Peptostreptococcus micros, S. sanguis II, and Fusobacterium nucleatum as the dominant organisms. Further, S. mitis and Gemella hæmolysans were generally found to be present. The enrichment cultures produced a wide variety of mainly cell-bound hydrolytic enzymes. This resulted in almost complete breakdown of salivary glycoproteins in the culture.The present experiments were aimed at studying the degradation of salivary glycoproteins by the oral microflora. To this end, S. sanguis I strain Ny476 and S. sanguis II (S. mitior) strain Ny581 were grown continuously in human-whole saliva. Under these conditions, the strains produced a variety of cell-associated hydrolytic activities, including glycosidases, exo- and endopeptidases, and esterases. S. sanguis II generally exhibited higher levels of enzyme activity than did S. sanguis I, in particular of neuraminidase that was produced only by S. sanguis II. In accordance, S. sanguis II had a higher cell yield and consumed a higher proportion of the sugars and sialic acid in the glycoproteins than did S. sanguis I. Interestinglv, S. sanguis I, which is devoid of neuraminidase, is known to have a lectin with specificity for sialic acid, whereas S. sanguis II has affinity for galactose residues in the glycoproteins. We propose that specific binding of glycoproteins by oral bacteria constitutes a mechanism t...

Effects of Fluoride and Chlorhexidine on the Microflora of Dental Root Surfaces and Progression of Root-surface Caries

The effects of fluoride and chlorhexidine varnishes on the microflora of dental root surfaces and on the progression of root-surface caries were studied. Forty-four patients, surgically treated for advanced periodontal disease, were distributed at random among three groups. All patients received a standardized preventive treatment. Furthermore, the dentition of the patients in the two experimental groups was treated, at three-month intervals, with chlorhexidine and fluoride varnish, respectively. Patients in the control group received no additional treatment. In the experimental groups, plaque samples were collected from selected sound and carious root surfaces at baseline and at three, six, and nine months after the onset of the study. The presence of root-surface caries was scored at baseline and after one year. In addition, the texture, depth, and color of the root-surface lesions were monitored. Mutans streptococci on root surfaces were suppressed significantly (p<0.05) during the whole experimental period in the chlorhexidine varnish group, but not in the fluoride varnish group. A non-significant increase in the number of Actinomyces viscosus/naeslundii was noted after treatment with chlorhexidine and fluoride varnish. The increase in the number of decayed and filled root surfaces after one year was significantly lower in the experimental groups than in the control group. After treatment with chlorhexidine varnish, significantly more initial root-surface lesions had hardened than in the other groups.

Comparative Recovery of Streptococcus mutans on Five Isolation Media, Including a New Simple Selective Medium

For the isolation of Streptococcus mutans, several selective media have been developed, of which Mitis-Salivarius Sucrose Bacitracin agar (MSB) is the most widely used (Gold et al., 1973). Recently, the Trypticase Yeast-Extract Cystine agar medium (TYC, de Stoppelaar et al., 1967) was modified into a selective medium for S. mutans, called Trypticase Yeast-Extract Cystine Sucrose Bacitracin (TYCSB, van Palenstein Helderman et al., 1983). The aim of this study was to compare the recovery of S. mutans from clinical samples on Mitis-Salivarius agar (MS), MSB, TYC, and TYCSB. Further, a new simple selective medium for S. mutans was introduced. This medium, called TSY20B, was supposed to have the same qualities as TYCSB, but its preparation is less laborious. One hundred eighty-five plaque and saliva samples from 37 subjects were plated on MS, MSB, TYC, and TYCSB, and 285 samples from 23 subjects were plated on TYCSB and TSY20B. All plates were incubated at 37°C in a 91% N2, 5% CO2, 4% H2 atmosphere for five days. The S. mutans counts on TYC and TYCSB were significantly higher than on MS or MSB by almost a factor of 10. Seventy-seven percent of the samples gave higher S. mutans counts on TYCSB than on MSB. Especially, samples with high S. mutans dlg numbers gave lower S. mutans counts on MSB. These data clearly indicate that MSB agar is inhibitory for S. mutans and should not be used. An additional advantage of TYCSB over MSB agar is the possibility of distinguishing S. mutans serotypes dlg from other serotypes. No significant difference was found between the recovery of S. mutans on TYCSB and its simplified version, TSY20B.

Decreased cariogenicity of a mutant of Streptococcus mutans

A strain of Streptococcus mutans was treated with a mutagenic agent. This resulted in isolation of a mutant which, compared to the original strain, had lost the ability to form sticky deposits on hard surfaces in sucrose medium. Apart from colonial morphology, the mutant had not changed in any other characteristic studied. In a 9-week experiment with SPF Syrian hamsters the average caries score induced by the mutant was significantly lower than that induced by the parent strain. In a 5-week experiment using germfree rats, the mutant had virtually lost its cariogenicity in sharp contrast to the original Strep, mutans. In all instances the implanted microorganisms could be recovered from the animals during all phases of the experiments.

Symbiosis of Streptococcus mutans and Veillonella alcalescens in mixed continuous cultures

Abstract The growth and metabolism of V. alcalescens OMZ 193 and Strep. mutans C67-1 were followed in a complex medium in pure and mixed continuous cultures. The fermentation products were assessed at several dilution rates. Strep. mutans formed ethanol, acetate and formate in addition to lactate. The ratio between these products was affected by the growth rate. The amount of ethanol and volatile fatty acids increased at decreasing growth rates. The fermentation of lactate by V. alcalescens yielded an equivalent amount of acid products. In mixed cultures both strains formed a food chain in the degradation of glucose. The symbiosis of Strep. mutans and V. alcalescens did not result in a reduction of the equivalent amount of acid produced per mole of glucose.

Lactobacilli, Mutans Streptococci and Dental-Caries - a Longitudinal-Study in 2-Year-Old Children up to the Age of 5 Years

A 3-year cohort study was carried out in 252 pre-school children for early identification of caries-active individuals. During this period information was collected about the acquisition of mutants streptococci and lactobacilli from the age of 2 till 5 years old. At baseline mutants streptococci were detected in 43% of the children while the detection frequency of lactobacilli was low (11.5%). On an individual level, numbers of colony-forming units of mutans streptococci and lactobacilli in plaque and saliva varied largely during the study period. The correlations between the numbers of lactobacilli and mutants streptococci in the saliva of the mother and the saliva and plaque of the child were low and never exceeded r = 0.22. Very low correlations (< r = 0.22) were also found between the numbers of mutans streptococci or lactobacilli and the diet in terms of the number of sugar intakes. Nevertheless, in children older than 2.5 years correlations between the clinical caries score and lactobacilli in saliva (range 0.31-0.62) and mutans streptococci in plaque or saliva (range 0.24-0.46) were highly significant (p < 0.01).

Enrichment of subgingival microflora on human serum leading to accumulation of Bacteroides species, Peptostreptococci and Fusobacteria

This study was undertaken to identify ecological factors that favour opportunistic pathogenic species in the subgingival microflora. In a first approach, human serum as a substitute for gingival exudate, was used for batch-wise enrichment of subgingival plaque. The microflora resulting after 5–6 enrichment steps consisted of black-pigmented and non-black-pigmented Bacteroides species, Peptostreptococcus micros and Fusobacterium nucleatum as the main organisms. It is noted that the same group of species was found to be enriched independent upon the origin of the subgingival plaque sample. It was suggested that these organisms are favoured by the increased flow of gingival exudate during inflammation.The consortium of organisms was capable of selective degradation of serum (glyco-)proteins. Four different types of degradation occurred. After a prolonged period of growth complete degradation of immunoglobulins, haptoglobin, transferrin and complement C3c was observed. Partial degradation of immunoglobulins, haptoglobin, transferrin, albumin, alpha1-antitrypsin and complement C3c and C4 was generally observed after 48 h of growth. Besides, immunoglobulin protease activity yielding Fc and Fab fragments was found. The consortium was also capable of consuming carbohydrate side-chains as indicated by an altered electrophoretic mobility of the serum glycoproteins.

Effects of Varnishes Containing Chlorhexidine on the Human Dental Plaque Flora

This study describes the effects of varnishes containing 0%, 10%, 20%, or 40% chlorhexidine diacetate on the microflora of human fissure dental plaque. Sandarac, a natural resin, was used as the varnish base. Ten subjects, each with at least four sound fissures harboring high levels of Streptococcus mutans, participated in the study. The fissures in each of the individuals were randomly assigned to four experimental groups, in each of which one of the varnishes was tested. The varnish treatment consisted of a single application of a small amount of varnish onto the fissures. Apart from the selected fissures, the rest of the dentition was left untreated. All chlorhexidine-containing varnishes selectively suppressed S. mutans in fissure plaque, and had no effect on total viable counts or on the numbers of Actinomyces viscosus/naeslundii and Streptococcus sanguis beyond one week. The extent of the suppression depended upon the concentration of chlorhexidine in the varnish, 40% chlorhexidine varnish giving the greatest suppression of S. mutans. At 22 weeks, after a single treatment with varnish containing 40% chlorhexidine, mean S. mutans counts were more than ten times lower than in the control or 10%chlorhexidine varnish group. At that time, S. mutans was still undetectable in five out of ten experimental fissures in this group. The results suggested that sandarac varnishes containing high concentrations of chlorhexidine can be used successfully for long-term suppression of S. mutans in dental fissures.

Synergistic Degradation of Mucin by Streptococcus oralis and Streptococcus sanguis in Mixed Chemostat Cultures

Oral streptococci can grow in mucin by utilizing the oligosaccharide chains as a source of carbohydrate. The degradation of the oligosaccharides by these species is accomplished by exoglycosidase activities. In this experiment, it was investigated whether strains from different species could cooperate in the release of sugars from the mucin oligosaccharide. To this end, Streptococcus sanguis Ny 584 and Streptococcus oralis strain Ny 586 were grown continuously in a chemically-defined medium, with pig gastric mucin as the growth-limiting source of carbohydrate. In pure cultures, strain Ny 586 attained approximately three-fold-higher cell densities than did strain Ny 584 in the mucin medium. This was in accordance with the observation that S. oralis Ny 586 exhibited fucosidase activity, as indicated by the presence of fucose in the culture fluid. In contrast, strain Ny 584 has no fucosidase activity against mucin, and therefore cannot attack fucose-ending oligosaccharide chains. Stable mixed cultures of the strains were obtained. It appeared that S. sanguis Ny 584 reached significantly higher cell densities in mixed cultures with S. oralis Ny 586 than in pure culture. Stimulation of the growth of strain Ny 584 was probably due to the generation of non-fucose-ending oligosaccharide chains by fucosidase from strain Ny 586. It is concluded that the synergistic degradation of oligosaccharides in glycoproteins is a potential factor influencing the streptococcal populations in the mouth.

Utilization of mucin by oral Streptococcus species

The ability of oral Streptococcus strains to utilize oligosaccharide chains in mucin as a source of carbohydrate was studied in batch cultures. Pig gastric mucin, as a substitute of human salivary mucin, was added to chemically defined medium containing no other carbohydrates. Strains of S. mitior attained the highest cell density, while mutans streptococci: S. mutans, S. sobrinus, S. rattus, grew very little in the medium with mucin. S. mitis, S. sanguis, and S. milleri in decreasing order, showed intermediate growth. Mucin break-down as measured by sugar analyses indicated that oligosaccharide chains were only partially degraded. Every strain produced one or more exoglycosidases potentially involved in hydrolysis of oligosaccharide. The enzyme activities occurred mainly associated with the cells, and very little activity was found in the culture fluids. The relationships between glycosidase activities and growth, or mucin degradation were not always clear.